Human milk oligosaccharides (HMOs) have become of great interest in the past few years due to their important functions in human development. To date, the structures of at least 115 HMOs have been determined (see Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1), and considerably more are probably present in human milk. The thirteen core structures identified to date, for the 115 HMOs, are listed in Table 1:
TABLE 1Core HMO structuresNoCore nameCore structure1lactose (Lac)Galβ1-4Glc2lacto-N-tetraose (LNT)Galβ1-3GlcNAcβ1-3Galβ1-4Glc3lacto-N-neotetraose (LNnT)Galβ1-4GlcNAcβ1-3Galβ1-4Glc4lacto-N-hexaose (LNH)Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc5lacto-N-neohexaose (LNnH)Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc6para-lacto-N-hexaose (para-LNH)Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc7para-lacto-N-neohexaose (para-Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcLNnH)8lacto-N-octaose (LNO)Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc9lacto-N-neooctaose (LNnO)Galβ1-4GlcNAcβ1-3(Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc10iso-lacto-N-octaose (iso-LNO)Galβ1-3GlcNAcβ1-3(Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc11para-lacto-N-octaose (para-LNO)Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc12lacto-N-neodecaose (LNnD)Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4Glc13lacto-N-decaose (LND)Galβ1-3GlcNAcβ1-3[Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4Glc
Low cost ways have been sought for making industrial quantities of as many as possible of the HMOs, so that their uses in nutritional and therapeutic formulations for infants, as well as as possibly children and adults, could be discovered, developed and exploited by researchers worldwide. A few HMOs have recently been chemically synthesized in high yields, for example, by hydrogenating their protected benzylated derivatives after removing other protecting groups from such protected derivatives and then crystallizing them from organic solvents. See WO 2010/115935 (2′-O-fucosyllactose or 2′-FL), WO 2011/100980 (lacto-N-neotetraose or LNnT), WO 2012/155916 (lacto-N-tetraose or LNT), WO 2011/100979 (6′-O-sialyllactose or 6′-SL) and WO 2012/007585 (various HMOs), and WO 2011/150939 (2′-FL polymorphs).
However, the relatively pure, crystalline HMOs, synthesized according to the above-mentioned methods may still be contaminated with small residues, i.e., about 1000 to 2000 ppm, but at least 100 ppm, of one or more of the following: i) toluene from the benzyl glycoside protecting group removed from their protected precursors by hydrogenolysis and ii) protic solvents such as C1-C6 alcohols used as the solvent or co-solvent with water in the hydrogenolysis step and/or used as the solvent or co-solvent in a subsequent crystallization or recrystallization of the HMOs. In order to use the HMOs in nutritional and therapeutic formulations for mammals, especially for humans, particularly for infants, it has been necessary to substantially reduce such residual contaminants, e.g., down to 500 ppm or less, preferably down to 100 ppm or less. Heretofore, this has required additional costly processing of the HMOs.
Accordingly, it is an object of the present invention to provide a method for removing, or reducing substantially the amount and/or concentration of organic solvent residues in and/or on HMOs, HMO precursors and blends, particularly blends of several HMOs and/or HMO precursors.
Moreover, crystalline HMOs have shown to be relatively unstable when stored for extended periods without refrigeration. They have tended to melt and thereby become sticky and form agglomerations.
Accordingly, it is also an object of the present invention to provide a method for enhancing the stability of HMOs, especially HMO blends, so that they can be stored for extended periods without refrigeration, for example at temperatures of up to 30° C. or even higher, preferably up to 40° C. or even higher.
Moreover, crystalline HMOs, as well as their precursors, and blends, have been relatively difficult to dissolve in water, so that they can be readily mixed with other active ingredients and suitable adjuvants for making liquid and powder, nutritional and therapeutic formulations. In addition, they have dissolved relatively slowly in the aqueous acid in the stomachs of mammals, particularly humans, quite particularly infants, which has reduced their bioavailability for the mammals.
Accordingly, it is a further object of the present invention to provide a method for enhancing the ability of HMOs, as well as their precursors, and blends, to dissolve in water and in the stomach acid of mammals.
Recently, CN 102154163 A disclosed spray-drying of an HMO, namely 3′-SL, produced by fermentation.